This application for a Mentored Clinical Scientist Development Award (K08) proposes a thorough investigation into the role of endogenous activators of the innate immune system in allospecific T cell activation. The candidate is an immunologist and transplant surgeon whose long-term goal is to prevent loss of transplanted organs due to immune rejection. This proposal will fulfill the educational objective of the development award by facilitating the expansion of the applicant's knowledge base into novel lines of inquiry requiring mentorship in these new areas. The expertise of the mentors assisting in this grant proposal will be essential to the successful completion of both the educational mission of the award as well as the performance of the proposed research plan that spans these areas. Thomas Coffman, MD will provide expertise in inflammation and mouse models of transplantation; Paul Noble, MD will provide expertise in glycobiology and has extensive experience investigating endogenous innate immune agonists; John Olson, MD, PhD will provide critical mentorship on balancing careers in surgery and research. Additionally, the candidate will participate in a rigorous career development plan as outlined in this application that will be instrumental in ensuring the successful transition of this candidate to being an independent researcher. PROJECT SUMMARY: With the discovery of Toll-like receptors (TLRs) there has been an increased appreciation of the critical role of innate immune pathways in regulating adaptive immune responses. It is now recognized that endogenous molecules derived from tissue injury can serve as TLR ligands. In the setting of transplantation, donor organs are subjected to many potential sources of sterile tissue injury, including: donor brain death, prolonged ischemia time, reperfusion injury, surgical trauma, and immunological injury. Importantly, each of these factors is associated with increased risk of graft rejection. How graft injury specifically contributes to graft rejection, however, is unknown. Based on preliminary data, this work hypothesizes that heparan sulfate (HS), an extracellular matrix polysaccharide released during injury, acts as a damage-associated molecular pattern (DAMP) molecule that activates TLR4 and can promote allospecific T cell activation. The proposed work will: 1) define the molecular pathways by which HS activates allospecific T cells, 2) determine how modulation of HS serum levels in a murine cardiac transplantation model affects the kinetics of graft rejection, and 3) define the role of hot and graft innate immune responses in allospecific T cell activation. This proposed work is innovative, in that it systematically investigates a new pathway of immune stimulation in the setting of sterile tissue injury that has implications for how organ grafts are preserved for transplantation, the detection of graft rejection, and the potential treatment of graft rejection. he approach uses novel transgenic mouse models to investigate donor and recipient contributions to innate immune stimulation and the activation of T cell subsets with direct and indirect allospecificity. PROJECT RELEVANCE: The proposed work is directly relevant to understanding fundamental mechanisms of immune activation by tissue injury and to developing therapeutic approaches for mitigating these responses in order to improve outcomes in organ transplantation. PROJECT NARRATIVE: The overall goal of this project is to understand the mechanism by which injury to organ grafts can activate the immune system of transplant recipients and render recipients more prone to rejecting their grafts. Activation of particular immune cell sensors, termed Toll-like receptors, seems to be critical in this process, and we will determine how heparan sulfate, a sulfated polysaccharide known to be released by tissues during injury, activates the immune system via these sensors. Understanding how injury to the organ graft can activate the immune system is important because it will illuminate targets for therapies aimed at improving the outcomes of transplant recipients.